TNF has been expected for use as an antitumor agent because it exerts an extremely strong antitumor effect on various tumor cells. However, TNF has not yet been successfully utilized as a pharmaceutical because of its excess side effect on living bodies due to its cytotoxic activity. When TNF is endogenously produced in a patient with a cancer or infectious disease, it may induce an inflammatory response and sometimes become an aggravating factor for such conditions. Therefore, development of a method to control TNF actions has been desired. As a method to control TNF activity, there has been a method for neutralizing TNF activity by administering antibodies, which bind to TNF, to living bodies. Pharmaceuticals containing such antibodies as effective ingredients have been already established. However, the above method may have a risk of canceling all beneficial biophylactic activities of TNF.
One TNF receptor having a molecular weight of 55 kilo daltons (hereinafter, it is called “TNF-R1”) and the other with 75 kilo daltons (hereinafter, it is called “TNF-R2”) are generally known as TNF receptors. Van Ostade X., Tavernier J., Prange T., and Fiers W. reported in “Localization of the active site of human tumor necrosis factor (hTNF) by mutational analysis”, The ENMO Journal, Vol. 10, No. 4, pp. 827-836, 1991, that alpha-type TNF (hereinafter, it is called “TNF-α”) of human origin, which dose not bind to mouse TNF-R2 and exerts a different biological effect differing from mouse TNF-α capable of binding to the both TNF receptors. Therefore, TNF mutant proteins selectively binding to either TNF-R1 or TNF-R2 would be expected to exert a different effect from that of conventional TNFs.
In order to create the TNF mutant proteins selectively binding to either TNF-R1 or TNF-R2, i.e., a receptor-specific TNF mutant protein, Van Ostade X et al. examined the binding sites in TNF-α to TNF-R1 or TNF-R2 by the mutational analysis as described in the above literature. As a result, they disclosed that mutagenesis at positions 29 to 34, 86 and 146 (amino acid residue numbers from the N-terminal of the amino acid sequence of TNF-α) enabled to weaken the binding affinity for TNF-R2, while mutagenesis at positions 143 to 145 enabled to weaken the binding affinity for TNF-R1. Particularly, they disclosed that TNF-α mutant protein, where the 32nd arginine (R) from the N-terminal was replaced with tryptophan (W), and the 86th serine (S) from the N-terminal was replaced with threonine (T), was extremely weakened in binding affinity for TNF-R2 while retaining the one for TNF-R1. Referring to Japanese Patent Publication Nos. 256395/94 and 285997/95; Zhang X M, Weber I and Chen M J, “Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship”, Journal of Biological Chemistry, Vol. 267, No. 33, pp. 24069-24075, 1992; and Loetscher H, Stueber D, Banner D, Mackay F and Lesslauer W, in “Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptor”, The Journal of Biological Chemistry, Vol. 268, No. 35, pp. 26350-26357, 1993, mutagenesis at positions 29 to 34, 81 to 89, and 143 to 147 (amino acid residue numbers from the N-terminal of TNF-α) may be hopeful to impart the receptor specificity to TNF mutant proteins. Receptor-specific TNF-α mutant proteins, retaining TNF activity as agonists, were prepared in the above literatures and might be expected as a TNF preparation with a potential TNF medical effect.
While, if TNF antagonist, which is a TNF mutant protein having a weak or no TNF activity, selectively binds to either of the two TNF receptors, it enables to allow an endogenously produced TNF to selectively bind to the remaining TNF receptor. However, the above literatures disclosed no TNF mutant protein having antagonistic activity specific to either of the TNF receptors. Therefore, receptor-specific TNF antagonist has been desired to develop.